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首页> 外文期刊>Journal of Molecular Biology >The basis for the super-repressor phenotypes of the AV77 and EK18 mutants of trp repressor.
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The basis for the super-repressor phenotypes of the AV77 and EK18 mutants of trp repressor.

机译：trp阻遏物的AV77和EK18突变体的超阻遏物表型的基础。

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The DNA-binding properties of two super-repressor mutants of the Escherichia coli trp repressor, EK18 and AV77, have been investigated using steady-state fluorescence anisotropy measurements, in order to further elucidate the basis for their super-repressor phenotypes. Several suggestions have been previously proposed as the basis for the super-repressor phenotype of EK18 and AV77. For the negative to positive charge change EK18 mutant, increased electrostatic interactions between the EK18 mutant and the operator and increased protein-protein interactions between EK18 dimers have been suggested as contributing to the super-repressor phenotype of this mutant. We show that EK18 dimers actually bind to wild-type and variant operator sequences with a decrease in apparent cooperativity and an increase in affinity, compared to WTTR dimers. Thus, the EK18 super-repressor phenotype is not due to increased cooperative binding between EK18 dimers. These results support the hypothesis that the super-repressor phenotype of EK18 arises from increased electrostatic interactions between the mutant and DNA. In the case of the AV77 mutant, weaker binding affinity of apo-AV77 to non-specific DNA, increased selectivity of binding of AV77 for the operator, and a higher population of folded functional AV77 dimers available to bind the operator under limiting L-Trp conditions in vivo, have been proposed for the super-repressor phenotype of this mutant. We show that like the EK18 mutant, apoAV77 binds with higher affinity to non-specific DNA compared to apo-WTTR and that the holo-AV77 mutant does not bind with higher selectivity to the operator, has had been previously proposed. We therefore conclude that the super-repressor phenotype of the AV77 mutant is due to an increase in the population of folded, functional AV77 dimers, under limiting L-Trp conditions in vivo. Copyright 2000 Academic Press.
机译：为了进一步阐明其超级阻遏物表型的基础，已使用稳态荧光各向异性测量研究了大肠杆菌trp阻遏物的两个超级阻遏物突变体EK18和AV77的DNA结合特性。先前已经提出了一些建议，作为EK18和AV77的超阻遏表型的基础。对于从负到正的电荷变化EK18突变体，已提出EK18突变体与操纵基因之间增加的静电相互作用以及EK18二聚体之间的蛋白质-蛋白质相互作用增加，这有助于该突变体的超阻遏表型。我们显示，与WTTR二聚体相比，EK18二聚体实际上与野生型和变异操纵基因序列结合，表观协同性降低，亲和力增加。因此，EK18超阻遏物表型不是由于EK18二聚体之间的协同结合增加。这些结果支持以下假设：EK18的超阻遏表型来自突变体与DNA之间增加的静电相互作用。在AV77突变体的情况下，载脂蛋白-AV77与非特异性DNA的结合亲和力较弱，对操纵基因的结合的选择性提高，在限制L-Trp的条件下，可以结合操纵基因的折叠功能性AV77二聚体的数量增加对于这种突变体的超阻遏表型，已经提出了体内条件。我们显示，像EK18突变体一样，与apo-WTTR相比，apoAV77与非特异性DNA的结合亲和力更高，而holo-AV77突变体与操作员的选择性更高，其结合力先前已被提出。因此，我们得出结论，在体内有限的L-Trp条件下，AV77突变体的超阻遏表型是由于折叠的功能性AV77二聚体的数量增加所致。版权所有2000学术出版社。

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